Abstract Glioblastoma (GBM) is the most common and malignant primary brain tumor with a median survival time of 12-15 months. Surgical resection followed by radiation and chemotherapy provides initial relief, but a subpopulation of tumor cells typically resists or evades therapy leading to tumor recurrence. GBM is characterized by a diffuse, infiltrative pattern of spread, with cells slowly crossing the hyaluronic acid (HA)-rich parenchyma towards vascular beds, where the linear structure of vasculature combined with local chemotactic and haptotactic gradients promote rapid, perivascular dissemination. While this process is well understood, the mechanisms that control the transition between intraparenchymal and perivascular invasion are not. The transmembrane receptor CD44 is a prime candidate for mediating this transition. CD44 is strongly enriched in the most invasive tumor cells and directly facilitates invasion by intracellular cytoskeletal binding and extracellular engagement of HA. Despite the acknowledged importance of CD44 as a mechanistic driver and potential therapeutic target, CD44-based motility, particularly in context of changing ECM cues, remains poorly understood.! The overall goal of this proposal is to investigate biophysical mechanisms through which CD44 channels HA-dependent signals to the cytoskeleton to regulate the transition between intraparenchymal and perivascular invasion. The first aim is to investigate the influence of CD44-cytoskeletal interactions on invasion using GBM culture models combined with motility assays, biomaterial platforms, and ex vivo brain slice models. The following hypotheses will be tested: 1) that the cytoskeletal binding domain of CD44 is important in 3D invasion through the parenchyma, but that this role diminishes as integrin binding sites increase in availability; 2) that CD44-cytoskeletal interactions slow cell migration along basal lamina interface by increasing interaction with the parenchyma. While initial studies will leverage existing invasion paradigms, a significant challenge in dissecting GBM invasion is that these standard platforms do not recapitulate the three-dimensional structure and architecture of extracellular matrix in the brain. Thus, the second aim is to engineer novel 3D models of the perivascular niche to study vascular homing, which will be used to test the hypothesis that CD44 synergizes with biophysical signals from endothelial cells to increase vascular homing and facilitate the transition to vascular invasion. This work will produce new insight into how CD44 acts through the cytoskeleton to drive GBM invasion and result in an innovative new perivascular niche model that will help the field investigate GBM invasion.